Xu Z H, Buelt M K, Banaszak L J, Bernlohr D A
Department of Biochemistry, Medical School, University of Minnesota, Minneapolis 55455.
J Biol Chem. 1991 Aug 5;266(22):14367-70.
The murine adipocyte lipid binding protein (ALBP/aP2) has been cloned and expressed in Escherichia coli, purified to homogeneity, biochemically characterized, and crystallized for x-ray diffraction study. In the cloning, the ALBP coding region was placed under control of the recA promoter and downstream of the phage T7 g-10 translation enhancer sequence. Nalidixic acid (50 micrograms/ml) induced the expression of ALBP 20-fold over that attained using the pT7 system previously reported (Chinander, L. L., and Bernlohr, D. A. (1989) J. Biol. Chem. 264, 19564-19572). Recombinant ALBP was purified to homogeneity using a combination of pH fractionation, gel filtration, and immobilized metal affinity chromatography. The fluorescent affinity ligand 12-(9-anthroyloxy)oleic acid bound to homogeneous ALBP with an apparent Kd of 0.5 microM. rALBP was devoid of endogenous fatty acid, and oleic acid inhibited cysteine 117 modification by 5,5' -dithiobis-(2-nitrobenzoic acid) indicating integrity of the binding domain. Recombinant ALBP was phosphorylated by the soluble kinase domain of the insulin receptor with a Vmax of 11 nmol.min.mg of kinase and an apparent Km of 270 microM. Purified protein was crystallized using the hanging drop method with seeding. Crystalline ALBP was orthorhombic with cell dimensions of a = 34.4 A, b = 54.8 A, and c = 76.3 A. The space group was P212121, and there was one molecule per asymmetric unit.
小鼠脂肪细胞脂质结合蛋白（ALBP/aP2）已被克隆，并在大肠杆菌中表达，纯化至同质，进行了生化特性鉴定，并结晶用于X射线衍射研究。在克隆过程中，ALBP编码区置于recA启动子控制之下，并位于噬菌体T7 g-10翻译增强子序列的下游。萘啶酸（50微克/毫升）诱导的ALBP表达比先前报道的使用pT7系统（Chinander, L. L., and Bernlohr, D. A. (1989) J. Biol. Chem. 264, 19564-19572）所达到的水平高20倍。重组ALBP通过pH分级分离、凝胶过滤和固定化金属亲和层析相结合的方法纯化至同质。荧光亲和配体12-(9-蒽氧基)油酸与同质ALBP结合，表观解离常数Kd为0.5微摩尔。重组ALBP不含内源性脂肪酸，油酸抑制5,5'-二硫代双-(2-硝基苯甲酸)对半胱氨酸117的修饰，表明结合域完整。重组ALBP被胰岛素受体的可溶性激酶结构域磷酸化，Vmax为11纳摩尔·分钟·毫克激酶，表观Km为270微摩尔。纯化的蛋白采用悬滴法加晶种进行结晶。结晶的ALBP为正交晶系，晶胞参数为a = 34.4埃，b = 54.8埃，c = 76.3埃。空间群为P212121，每个不对称单元有一个分子。
